The goals of this project are to define the biochemical and cellular events in the processing of protein antigens by APC and the selection of peptides by the class II MHC molecules. The applicant will continue examining peptide selection from model protein antigens like lysozyme (HEL), ribonuclease and hemoglobin. He will investigate where the protein antigen goes in the APC, how it is initially fragmented, how the MHC binds the early catabolites, whether the early catabolites bound to MHC are subsequently trimmed, and how the peptide MHC complex is handled by the APC. All these events translate in the selection of dominant peptide sequences presented at the plasma membrane of APC. Initial studies on the biochemistry of peptide from HEL strongly justify a biochemical approach. For example, the dominance by a single epitope is striking and, moreover, the dominant epitope is presented as nested peptides with extensions not previously predicted by immunological assays. Such nested peptides have important chemical and biological consequences. Aim 1 will continue to sample I-Ak and I-Ek molecules from large scale cultures of APC with antigens in a biochemical effort to define the distribution of the selected peptides. Issues that are relevant are the amounts of protein, the kind of APC, the time of examination, and the presence of dominant and sub-dominant peptides. This aim is based on technologies that are all in place and include large scale growth of APC, MHC isolation, HPLC fractionation of eluates, sequencing of peptides and mass spectrometry examination. The second aim is to engineer mutations in the HEL biochemical basis for the peptide selection. The HEL molecule will be presented as a fusion protein linked to either part of the Ld or the Cathepsin D molecule. The applicant will investigate whether changes in the core residues of the dominant epitopes influence whether minor epitopes become apparent, which may explain the biochemical basis of dominant and sub-dominant epitopes. It also will be investigated whether changes in the residues that flank the core peptide are important in peptide selection, addressing the issues of enzyme specificity and aminopeptidases. Finally, by using a combination of cellular approaches and subcellular fractionation, the cellular dynamics and fate of different peptide-MHC complexes will be examined.